Previous studies from our lab have provided evidence that transcellular NaCl reabsorption in the proximal tubule arises from parallel operation of apical membrane Na-H and Cl-base exchange processes. During the past project period, we were successful in identifying CFEX (SLC26A6) as the transporter that likely accounts for apical membrane Cl-base exchange in the proximal tubule. NHE3 has been identified as the Na-H exchanger isoform responsible for the majority of apical membrane Na-H exchanger activity in the proximal tubule. The general goal of the proposed project is to understand the mechanisms underlying regulation of these two key proximal tubule transporters. One set of studies will evaluate the mechanisms of regulation of SLC26A6 heterologously expressed in Xenopus oocytes and endogenously expressed in mouse proximal tubule. We will specifically assess the role of protein kinase C in regulating SLC26A6 activity as suggested by preliminary data. We will then evaluate whether SLC26A6-mediated Cl absorption in the proximal tubule is regulated by acute and chronic stimuli that are known to regulate NHE3 activity, and we will examine the role of PKC in the regulation that is observed. Mutagenesis studies will be carried out to address the role of SLC26A6 phosphorylation, and to identify domains involved in its possible regulation by membrane trafficking. Another set of studies will generate and purify antibodies that are specifically reactive with NHE3 phosphorylated at serine residues shown in transfecfion studies to be critical for regulation of NHE3 by PKA. We will use these antibodies to assess the state of phosphorylation of these serine residues in endogenous NHE3 in native proximal tubule in response to acute and chronic physiologic stimuli that alter NHE3 activity. We will determine whether in response to these stimuli there is a change in the level of expression and/or the subcellular distribution of NHE3 phosphorylated at these residues. Taken together, these two sets of studies will provide new insight into the mechanisms of regulation of NHE3 and SLC26A6. Differential regulation of the activities of NHE3 and SLC26A6 would allow selective regulation of the rates of NaHCO3 reabsorption and NaCl reabsorption as appropriate to achieve acid-base and extracellular volume (NaCl) homeostasis, respectively.